This invention is in the field of peptide chemistry, in particular relating to methods for forming amide bonds in water useful in the synthesis of peptides and proteins in biological systems and also in the synthesis of derivatized peptides or proteins in aqueous solutions.
New methods are facilitating the total chemical synthesis of proteins. For historical references, see: Merrifield, R. B. Science 1984, 232, 341-347; Kent, S. B. Annu., Rev. Biochem. 1988, 57, 957-989; Kaiser, E. T. Acc. Chem. Res. 1989, 22, 47-54. In particular, Kent and others have developed a means to stitch together two unprotected peptides in aqueous solution called “native chemical ligation.” Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Science 1994, 266, 776-779. For important precedents, see: Wieland, T.; Bikelmann, E.; Bauer, L.; Lang, H. U.; Lau, H. Liebigs Ann. Chem. 1953, 583, 129-149; Kemp, D. S.; Galakatos, N. G. J. Org. Chem. 1986, 51, 1821-1829.
In native chemical ligation the thiolate of an N-terminal cystein residue in one peptide attacks the carbon of a C-terminal thioester in another peptide to produce, ultimately, an amide bond between the two peptides. This ligation method was discovered when the reaction of ValSPh and CysOH in aqueous buffer was shown to yield the dipeptide: ValCysOh (Wieland, T.; Bikelmann, E.; Bauer, L.; Lang, H. U.; Lau, H. Liebigs Ann. Chem. 1953, 583, 129-149).
Recently, Muir and others have expanded the utility of native chemical ligation by demonstrating that the thioester fragment can be produced readily with recombinant DNA (rDNA) techniques. Muir, T. W.; Sondhi, D.; Cole, P. A. Proc. Natl. Acad. Sci. U.S.A.; 1998, 9, 6705-6710; Evans, Jr., T. C.; Benner, J.; Xu, M.-Q. Protein Sci. 1998, 7, 2256-2264; Ayers, B.; Blaschke, U. K.; Camarero, J. A.; Cotton, G. J.; Holford, M.; Muir, T. W. Biopolymers 2000, 51, 343-354. For reviews, see: Holford, M.; Muir, T. W. Structure 1998, 15, 951-956; Cotton, G. J.; Muir, T. W. Chem. Biol. 1999, 6, R247-R256. Evans, Jr.; T. C.; Xu, M.-Q. Biopolymers 2000, 51, 333-342. This type of “native chemical ligation” has been designated “protein ligation”
Although native chemical ligation is useful in some systems, it has severe limitations. The method relies on the formation of a peptide bond to a cysteine residue. Creating this type of linkage is not always possible, as cystein comprises only 1.7% of the residues in globular proteins. Hence, most proteins cannot be prepared by a method that requires peptides to be coupled only at a cysteine residue.
Offer and Dawson have recently described a peptide ligation method that does not require the presence of cysteine. (Offer, J.; Dawson, P. E. Org. Lett. 2000, 2, 23-26). In their method, a peptide bond is formed from a thioester and mercaptobenzylamine. Though effective, this method necessarily leaves o-mercaptobenzylamine in the ligation product.
In the well-known Staudinger reaction a phosphine is used to reduce an azide to an amine:PR3+N3R′+H2O→O═PR3+H2NR′+N2(g)(Staudinger, H.; Meyer, J. Helv. Chim. Acta. 1919, 2, 635-646. For reviews, see: Gololobob, Yu. G.; Zhmurova, I. N.; Kasukhin, L. F. Tetrahedron 1981, 37, 437-472; Gololobov, Yu. G.; Kasukhin, L. F. Tetrahedron 1992, 48, 1353-1406). The intermediate in the reaction is an iminophosphorane (R″3P+—−NR), which has a nucleophilic nitrogen.
Recently, Saxon et al. have reported a modification of the Staudinger ligation to form an amide from an azide using a phosphine reagent. (Saxon, E.; Armstrong, J. I.; Bertozzi, C. R.; Org. Lett. 2000, 2, 2141-2143). The phosphine reagents:
when reacted with an azido nucleosides are reported to result in the formation of an amide by acryl group transfer. The ligation is called traceless because no portion of the phosphine reagent other that the acyl group remains in the product. The authors also report that the reaction of a phosphinothioester:
with the same azido nucleoside results initially in aza-ylide hydrolysis rather that acyl transfer. The observation of amide products after several days in characterized as the probably result of reaction of amine hydrolysis products with the thioester. The authors indicate that the phosphinothioester employed is not “amenable” to the reaction.
Recently, a strategy has been developed for protein assembly utilizing the Staudinger reaction. Nilsson, B. L; Kiessling, L. L.; Raines, R. T.; Org. Lett. 2000, 2, 1939-1941; Nilsson, B. L.; Kiessling, L. L.; Raines, R. T.; Org. Lett. 2001, 3, 9-12; Kohn, M.; Breinbauer, R.; Angew. Chem. Int. Ed. 2004, 43, 3106-3116. This traceless Staudinger ligation proceeds through an azide reduction by a phosphine via an iminophosphorane intermediate. This reaction is extremely useful as it can mediate the nonengrammic ligation of peptides at virtually any residue. Soellner, M. B.; Tam, A.; Raines, R. T. J. Org. Chem. 2006, 71, 9824-9830. The iminophosphorane can be acylated to yield an amine. The proposed mechanism is shown in Scheme 1.

Although this reaction is extremely useful, it requires the use of organic solvents and therefore has limited use in biological environments. U.S. Pat. Nos. 6,972,320; 6,974,884; 7,256,259 and 7,317,129 relate to this “traceless” method for formation of an amide bond between a phosphinothioester and an azide as well as to reagents useful in such methods. More specifically U.S. Pat. No. 7,256,259 relates to a method for covalent ligation of molecules to surfaces via amide bond formation. U.S. Pat. Nos. 6,974,884 and 7,317,129 relate to methods for synthesis of phosphinothiol reagents and reagents useful in the formation of amide bonds and for peptide ligation. The patent also relates to phosphine-borane complexes useful as reagents in such methods.